These studies are designed to define the molecular lesions affecting Beta globin production in patients with homozygous Beta thalassemia. During the past year we have completed characterization of a defective Beta globin gene in which a single nucleotide substitution T replacing A in the third position of codon 24 activates an alternative splice site. Codon 24 of both the normal gene (GGT) and the Beta+ thalassemia kgene (GGA) encodes glycine. Activation of the alternate splice site results in incorrect splicing of 80% of the Beta globin gene transcript; these incorrectly spliced molecules are apparently subjected to intranuclear degradation since the minority that reach the cytoplasm are stable. Analysis of a second Beta thalassemia gene characterized by a premature stop codon in position 39 indicates that such defective RNA molecules are also subjected to intranuclear destruction. There is an 8-10-fold reduction in Beta mRNA concentration in nuclei as well as cytopolasm and that minority of RNA that reaches the cytoplasm has normal stability. Function of these cloned Beta thalassemia genes was studied in monkey kidney cells by using plasmid expression vectors. As a model for thalassemia we have studied the Beta globin gene in the erythroleukemia cell line, K562 in collaboration with colleagues in the LCB in NIADDK. RNA mapping studies showed complete lack of Beta globin expression in these cells despite significant expression of the w gene. Restriction endonuclease analysis revealed two alleles at a polymorphic Ava II site in the Beta globin gene indicating that these cells contain two distinct non-functioning genes. One of these was cloned and was shown to function normally in the COS cell expression assay. Thus hypofunction of the Beta globin genes in K562 cells does not reflect defective gene structure.